Thermodynamic data are reported revealing that pseudouridine (Ψ) can stabilize RNA duplexes when replacing U and forming Ψ-A, Ψ-G, Ψ-U and Ψ-C pairs. Stabilization is dependent on type of base pair, position of Ψ within the RNA duplex, and type and orientation of adjacent Watson–Crick pairs. NMR spectra demonstrate that for internal Ψ-A, Ψ-G and Ψ-U pairs, the N3 imino proton is hydrogen bonded to the opposite strand nucleotide and the N1 imino proton may also be hydrogen bonded. CD spectra show that general A-helix structure is preserved, but there is some shifting of peaks and changing of intensities. Ψ has two hydrogen donors (N1 and N3 imino protons) and two hydrogen bond acceptors because the glycosidic bond is C-C rather than C-N as in uridine. This greater structural potential may allow Ψ to behave as a kind of structurally driven universal base because it can enhance stability relative to U when paired with A, G, U or C inside a double helix. These structural and thermodynamic properties may contribute to the biological functions of Ψ.